RNA interference (RNAi) is an evolutionarily conserved, sequence specific mechanism triggered by double stranded RNA (dsRNA) that induces degradation of complementary target single stranded mRNA and “silencing” of the corresponding translated sequences (McManus and Sharp, Nature Rev. Genet. 2002, 3:737; Mello and Donte, Nature 2004, 431:338; Meister and Tuschl, Nature 2004, 431:343; Sen and Blau, FASEB J. 2006, 20:1293).
Exploiting this mechanism has yielded a powerful tool to unravel the function and significance of hitherto unknown or uncharacterized genes in in vitro experiments (Hannon and Rossi, Nature 2004, 431:371; Westbrook et al., Cold Spring Harb Symp Quant Biol. 2005, 70:435): RNAi can be used to down-regulate or silence the transcription and translation of a gene product of interest; where said gene product is unknown or uncharacterized, the development of a certain phenotype can be used to determine the function and/or significance of the gene product. Great potential is also seen in harnessing the underlying cellular mechanisms for the therapy of human disease (Zhou et al., Curr Top Med. Chem. 2006, 6:901): where said gene product is in any way associated with a disease or disorder by way of its overabundance, its down-regulation may be used in the prevention and/or therapy of the disease or disorder.
The triggering of RNAi by dsRNA requires the dsRNA to be localized in the cytoplasm and/or nucleus of the cell in which the target gene is to be silenced. To this end, the dsRNA may be introduced directly into the cell, e.g., by bringing the cells into contact with the dsRNA, whereupon the dsRNA is actively or passively internalized. Therein, the dsRNA may be large, e.g., comprising 100, 200, 400 or more base pairs. A large dsRNA will be processed in mammals by an RNAse III-like enzyme commonly called Dicer to smaller fragments of 21 to 23 base pairs. Alternatively, the dsRNA may be small, e.g., of the size of the Dicer products (dsRNAs of this size, e.g., having not more than 30 base pairs, are in the art often referred to as short interfering RNAs, or siRNAs). The small dsRNAs, be they a product of Dicer activity or directly introduced, are subsequently unwound by, and one strand of the small dsRNA is incorporated into, a protein complex termed RISC(RNA induced silencing complex). RISC then proceeds to cleave mRNAs having a sequence complementary to the RNA strand that was incorporated into RISC (Meister and Tuschl, Nature 2004, 431:343).
In order to harness RNAi for any purpose in vitro and/or in vivo, a nucleic acid molecule must somehow be introduced into a cell, preferably into a cell that forms part of a living organism, such as a mammal or a human. If RNA interference is to live up to its potential, the process of introducing the nucleic acid molecule should disrupt the natural functions of the cell as little as possible, particularly where the cell is part of a living organism. This problem is shared by, for example, many procedures in genetic engineering, as well as gene therapy. Numerous solutions have been proposed, none of which is so far fully satisfactory.
The liver is one particularly attractive target for therapeutic intervention for a number of reasons: a) it plays a central role in many vital functions of the human body, b) it is the first pass organ for many substances absorbed from the gut and receives a large part of cardiac output, c) it is involved in many diseases and unwanted conditions with high prevalence in humans, e.g., Alagille syndrome, alcoholic liver disease, alpha-1-antitrypsin deficiency, Budd-Chiari syndrome, biliary atresia, Byler disease, dyslipidemias, Carol i-disease, Crigler-Najjar Syndrome, Dubin-Johnson Syndrome, fatty liver, galactosemia, Gilbert syndrome, glycogen storage disease 1, hemangioma, hemochromatosis, hepatitis of viral or autoimmune etiology, liver cancer, liver fibrosis and cirrhosis, porphyria cutanea tarda, erythrohepatic protoporphyria, Rotor syndrome, sclerosing cholangitis, or Wilson disease.
In the development of a treatment of hepatic diseases and conditions, it would be advantageous to have the capability to specifically target the cells of the liver with a therapeutic agent, e.g., an RNAi agent.
One approach documented in the literature has been conjugating the nucleic acid to a cholesterol moiety (Soutschek, J., et al., Nature 2004, 432:173-178), wherein the target gene was ApoB. However, the nucleic acid showed inhibition of ApoB not exclusively in the liver, but also in the gut of experimental animals. For an antisense oligodesoxynucleotide (ODN), adding a second cholesteryl moiety was effective in directing uptake of up to nearly 90% of a certain dose of the ODN to the liver (Bijsterbosch, M. K., et al., J. Pharmacol. Exp. Ther. 2002; 302:619).
Alternatively, a number of authors have proposed conjugating various molecular species, including ODN, to ligand moieties, e.g., via a variety of linkers, which bind the asialoglycoprotein receptor, to enhance hepatic uptake (Wu, G. Y., Wu, C. H., J. Biol. Chem. 1988, 263:14621; Biessen, E. A., et al., J. Med. Chem. 1995, 38:1538; Biessen, E. A. L., et al., Biochem. J. 1999, 340:783; Joziasse, D. H., et al., Eur. J. Biochem. 2000, 267:6501; Rump, E. T., et al., Biochem. Pharmacol. 2000; 59:1407; Biessen, E. A., Methods Enzymol. 2000, 314:324; Rensen, P. C. N., et al., J. Biol. Chem. 2001, 276:37577; Rossenberg, S. M. W., et al., J. Biol. Chem. 2002, 277:45803). The asialoglycoprotein receptor (ASGPR) is a transmembrane glycoprotein (42 kDa) which mediates binding, internalization and degradation of extracellular glycoproteins that have exposed terminal galactose residues. The receptor is expressed on the surface of hepatocytes, and only of hepatocytes, in a polar manner, i.e., it is present on the sinusoidal and lateral plasma membranes, but not on the bile canalicular membrane. The mammalian hepatic ASGPR mediates the endocytosis and degradation of serum proteins from which terminal sialic residues have been removed. The exclusive localization of the ASGPR to the liver, as well as its natural function in transporting comparatively large molecules into the hepatocyte, make it an attractive option for a mediator of liver cell targeting of therapeutic substances.
The available nucleic acid delivery systems are not yet satisfactory in terms of safety and/or efficiency for their utilization in in vitro experimental applications and/or human diagnosis and therapy, and require further optimization.
The technical problem underlying the present invention is the provision of improved methods and means for the delivery into cells of nucleic acid molecules, and preferably of RNAi agents, which are useful in vitro and in vivo, preferably for human therapy. This problem has been solved by the provision of the embodiments as characterized herein below and in the claims.